Stabilized Cyclopropane Analogs of the Splicing Inhibitor FD-895

Aug 6, 2013 - A Carbohydrate-Derived Splice Modulator. Sachin Dhar , James J. La Clair , Brian León , Justin C. Hammons , Zhe Yu , Manoj K. Kashyap ,...
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Stabilized Cyclopropane Analogs of the Splicing Inhibitor FD-895 Reymundo Villa,† Manoj Kumar Kashyap,‡ Deepak Kumar,‡ Thomas J. Kipps,‡,§ Januario E. Castro,‡,§ James J. La Clair,† and Michael D. Burkart*,† †

Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, United States ‡ Moores Cancer Center and §Department of Medicine, University of California San Diego, La Jolla, California 92093-0820, United States S Supporting Information *

ABSTRACT: Targeting the spliceosome with small molecule inhibitors provides a new avenue to target cancer by intercepting alternate splicing pathways. Although our understanding of alternate mRNA splicing remains poorly understood, it provides an escape pathway for many cancers resistant to current therapeutics. These findings have encouraged recent academic and industrial efforts to develop natural product spliceosome inhibitors, including FD-895 (1a), pladienolide B (1b), and pladienolide D (1c), into next-generation anticancer drugs. The present study describes the application of semisynthesis and total synthesis to reveal key structure− activity relationships for the spliceosome inhibition by 1a. This information is applied to deliver new analogs with improved stability and potent activity at inhibiting splicing in patient derived cell lines.

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INTRODUCTION Given the successful clinical advance of polyketide natural product analogs for treatment of diverse cancers,1 FD-895 (1a)2 and related pladienolides B (1b) and D (1c)3,4 have been the subject of considerable synthetic,5−11 biosynthetic,12−14 and mode of action (MOA) studies. The latter MOA studies identified15 and validated16 splicing factor SF3b as the primary target of 1b.17 In parallel efforts, SF3b has also been shown to be targeted by spliceostatin A (2, Figure 1c), an analog of the natural product FD901464.18−20 In 2006, Eisai Co. Ltd. launched clinical trials (NCT00459823) with a carbamate analog of 1b, E7101 (1d, Figure 1), for application in solid tumors.15,17 Concomitantly, we focused our efforts on a combination of semisynthetic and total synthetic methods to identify next-generation leads for further clinical examination. Since this effort, ongoing studies in our2,3 and other laboratories11,19,20 have confirmed the importance of small molecule regulators of splicing and the need for next-generation synthetic derivatives for clinical evaluation.21,22

Figure 1. Structures of 1a (FD-895), 1b (pladienolide B), 1c (pladienolide D), the clinical entry 1d (E7107), and the related spliceosome-targeting natural product derivative spliceostatin (2).

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RESULTS AND DISCUSSION Our efforts began with a detailed evaluation of the stability of 1a. As shown in Figure 2, we identified that 1a undergoes hydrolysis under neutral conditions to afford a mixture of acids 3a−3c.6 As determined by NMR monitoring (Figure 3), this reaction occurs with the formation of 3a, 3b, and 3c in an approximated 3:2:5 ratio (Figures 3 and 4a), suggesting the hydrolysis occurs via nonselective addition of water to an incipient allylic cation. While not unexpected, decomposition begins within 7 h in aqueous media. This process would be © 2013 American Chemical Society

expected to be much faster in serum or tissue, in which the combination of pH changes and enzymatic activity could accelerate the process. The hydrolyzed products collected at 216 h, a mixture of 3a−3c demonstrate significant loss of activity, with an IC50 value of 11.2 ± 1.4 μM (Table 1), over 1000-fold less active, in HCT-116 cells.23,24 On the basis of this Received: June 11, 2013 Published: August 6, 2013 6576

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Figure 2. In aqueous media, both natural and synthetic FD-895 (1a) undergo hydrolysis, resulting in acids 3a−3c. Figure 4. LC−HRMS traces depicting the products from incubating samples of (a) 10 mM 1a in D2O/DMSO-d6 (10:1); (b) 10 mM 6c in D2O/DMSO-d6 (10:1); (c) 10 mM 7a in D2O/DMSO-d6 (10:1) at 37 °C for 143 h. The plot in part a corresponds to the NMR point at 143 h in Figure 3. Hydrolysis of 1a, including acids 3a−3c, while derivatives 1e and cyclopropane 7a remained stable. Exact masses [M + Na]+ are provided for each peak.

Table 1. Growth Inhibition Data IC50 (nM) HCT-116 1a 1b 3a−3c (mixture) 4a 4b 4c 4d 5a 5d 6a 6b 6c 7a 7b

a

23.5 ± 0.2 24.8 ± 0.4 11 200 ± 1400 >50 000 >50 000 >50 000 >50 000 542.0 ± 12.1 142.2 ± 6.3 3.7 ± 0.2 23.0 ± 1.2 0.8 ± 0.1 42.9 ± 1.3 82.3 ± 3.2

CLL 2.1 ± 0.1 30.5 ± 0.2 142.0 ± 0.3

108.9 ± 0.2 >50 000 102.9 ± 0.1

a

IC50 values were collected using the MTT assay, and are presented as an average of 3−5 repetitions. bIC50 values were calculated by GraphPad Prism v5.0 to determine the concentration to induce 50% SIA using at least three different concentrations, as noted in the Experimental Procedures.

Figure 3. NMR monitor of the hydrolytic decomposition of 10 mM 1a in D2O/DMSO-d6 (10:1) at 37 °C. Hydrolysis occurs as marked by a shift of the C11 proton (large to small circles). Assignments of 3a, 3b, and 3c were made on the basis of the coupling constant and gCOSY analyses.

ester led to >100-fold loss in activity, suggesting that modifications at C7, as in E7107 (1d), could prove problematic. A second set of SAR data can be derived from analogs prepared in our synthetic campaign. Early efforts in our laboratories provided access to core-modified analogs 4a−4d (Figure 5).5 All four of these analogs proved inactive, returning IC50 values >50 μM when screened against the HCT-116 cell line using the MTT assay (Table 1).23,24 This observation was further confirmed by recent studies from the Webb laboratory, which demonstrated that analogs such as 4e lack activity.11 These studies also identified a TBS ether 4f that partially

observation, we turned our efforts to identifying analogs with improved hydrolytic stability. Although synthetic access has been established,5−11 a detailed understanding of the structure−activity relationship (SAR) within the family of macrolides encompassing 1a−1c has yet to be completed. The first piece of the SAR data was derived from probe development efforts associated with the MOA investigation.15 In these studies, linkers applied at the C7 6577

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Figure 5. Synthetic analogs 4a−4d proved to be devoid of activity (see Table 1), indicating that gross modifications to the core would not be tolerated. This was confirmed by comparable analogs, such as 4e. Other analogs, such as TBS-ether 4f, displayed modest activity.10

rescued activity, implying a need for a hydrophobic group at C21. Additional activity data were also obtained directly from 1a. Peracetylation of 1a afforded 5a (Figure 6) in excellent yield. In Figure 7. Structures of the C16−C17-isomers 6a−6c and C18−C19 cyclopropanes 7a and 7b.

conditions applied to 1a (as shown in Figure 2) indicated that 6c was more resistant to hydrolysis, with lack of the corresponding acid products appearing under the same conditions (data from sampling at 143 h is provided in Figure 4b). To further address stability, we then turned to examine the replacement of the epoxide moiety, which is commonly associated with toxicity, off-target reactivity, and instability.26 We chose to prepare cyclopropanes 7a and 7b, which share the stereochemical assignment at C16 and C17 with 6c and 6b, respectively (Figure 7). Synthesis of these materials began with allyl alcohol 8 (Scheme 1), a central component prepared on the multigram scale for the synthesis of 1a.6 Using a Charette cyclopropanation,27 we were able to obtain cyclopropanol 9 as a single diasteromer. Oxidation with Dess−Martin periodinane afforded aldehyde 10 in 66% yield from 8. Analysis of the 1H− NMR spectrum of 10 confirmed the stereochemistry as shown by coupling constants of JH18−H19 = 4.4 Hz (syn), JH19−H20 = 9.3 Hz (anti), JH20−H21 = 4.1 Hz (syn). Aldehyde 10 served as a central building block to intersect our established synthetic route to 1a.6 Using methods developed by Marshall,25 addition of allenylstannane 12 to aldehyde 10 afforded major isomer 13a along with minor isomer 13b. Fortunately, these materials were chromatographically separable. We then converted 13a and 13b to their respective analogs 7a and 7b of 1a using a rapid two-step process. As shown in Scheme 1, alkyne 13a was converted to vinylstannane 14a, which was quickly purified and coupled directly to core 15 (compound 14a was stable; however, we found improved yields when conducting a rapid flash purification and coupling this material directly to 15), delivering 7a in 23% overall yield from 8. Comparable methods provide isomer 7b in 16% from 8.

Figure 6. Semisynthetic analogs 5a−5d also failed to enhance the activity of 1a. Reagents and conditions: (a) Ac2O, DMAP, pyridine, 82%; (b) TESCl, imidazole, DMAP, THF, 8 h, rt, 87%; (c) Ac2O, DMAP, pyridine, 75%; (d) TBAF, wet THF, 16 h, 52%.

addition, 1a was selectively converted to the C3 triethylsilyl ether, which then could be converted to bisacetate 5d in a stepwise fashion. From this effort, we found that acetates 5a and 5d displayed reduced activity (see Table 1 and NCI-60 data provided within the Supporting Information). Recently, we reported the total synthesis6 of 1a and applied these methods to access analogs that were not readily prepared from 1a. Using the same intermediates used in our preparation of 1a, we were able to apply the allenylstannane chemistry of Marshall25 to prepare each of the C16−C17 isomers 6a−6c (Figure 7). Cytotoxicity analyses via the MTT assay23,24 indicated that all four isomers 1a and 6a−6c displayed high picomolar to low nanomolar activity when screened against the HCT-116 tumor cell line (see Table 1). Isomers with the natural R configuration at C16 were less active than those with S. Additional activity was also observed when C17 was left in its natural R configuration. Isomer 6c was nearly 25 times more active than 1a. Most importantly, application of the hydrolysis 6578

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Scheme 1. Synthesis of Cyclopropane Analogs 7a and 7b.a

Figure 8. RT-PCR analysis of splicing inhibitory activity. Splicing activity was determined by monitoring the increase of the unspliced DNAJB1 gene as compared with GAPDH (a housekeeping gene). (a) Compound screening identified 1a as the most potent inhibitor with derivatives 6a and 7a, also imparting splicing inhibition. Percent SIA denotes the percentage of apoptotic induction by this material. (b) Splicing ability correlates with the concentration of 7a. Controls are indicated by the following: (−) negative control or no compound added, (G) genomic DNA, and (F) fludarabine. Spliced (S) and unspliced (U) forms of DNAJB1 are shown. *A mixture of 3a−3c was obtained from the hydrolysis of 1a after 143 h at 37 °C in H2O/ DMSO (10:1). Acids 3a−3c were tested as a mixture.

dehydrogenase (GAPDH, a control housekeeping gene). Subsequent analyses (Figure 8b) indicated that although 7a inhibited splicing, the activity of 7a activity was lower than that of 1a and 1b. This finding, however, is quite remarkable. All known spliceosome inhibitors in the pladienolide and spliceostatin families, including hybrid analogs, have contained epoxide moieties considered essential for activity.11,19,20 However, analog 7a indicates that the epoxide moiety is not essential for spliceosome activity in 1a, offering the promise of improved pharmacophores for potential therapeutic use.

a Reagents and conditions: (a) 11, Zn, CH2I2, DME, −10 °C to rt, 83%; (b) Dess−Martin periodinane, CH2Cl2, 0 °C to rt, 80%; (c) 12, BF3·Et2O, CH2Cl2, −78 °C to rt, 80% of a 3:2 mixture of chromatographically separable 13a/13b; (d) PdCl2(PPh3)2, (nBu)3SnH, THF, rt; (e) Pd2(dba)3, AsPh3, LiCl, NMP, 73% for 7a over two steps from 13a and 80% for 7b from 13b.

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We then turned to evaluate the biological activity of synthesized compounds. When interpreting our NCI-60 cell screening data of select analogs of 1a (see example data set provided in the Supporting Information), we identified enhanced potency of several analogs in leukemia cell lines. We then focused our program on leads that delivered maximum potency in patient-derived chronic lymphocytic leukemia (CLL) cells.28 Using the induction of apoptosis as a marker, we determined that 1a displayed an IC50 value of 2.1 ± 0.1 nM (Table 1). The C16−C17 analog 6b lacked activity (IC50 value > 50 μM), whereas 6a displayed reduced activity with an IC50 value of 108.0 ± 0.2 nM. Most importantly, the cyclopropane derivative 7a retained activity delivering an IC50 value of 102.9 ± 0.1 nM (Table 1). We then implemented an RT-PCR gel-shift assay to confirm their splicing inhibitory activity. As shown in Figure 8, we could correlate the splicing activity of 1a with only the nonhydrolyzed natural product, as isolated hydrolyzed material proved inactive in this assay (see Figure 8a, 3a−3c vs 1a). We also learned that other analogs also inhibited the splicing of DNAJB1, DnaJ (Hsp40) homologue subfamily B member 1, but not by F-AraA (negative drug control) or glyceraldehyde 3-phosphate

CONCLUSION We have investigated the instability of 1a, which leads to 103 fold decrease in activity, followed by a SAR study to identify stabilized, active analogs. This included synthesis of cyclopropane derivatives of 1a and compounds 7a and 7b. Compound 7a displayed improved stability and effectively mimicked the apoptotic and splicing activity of 1a. Although the activity of 7a was less than 1a, its increase in stability provides a key next step in advancing stable materials for in vivo applications. Furthermore, this study demonstrates that labile functional groups, such as the C18−C19 epoxide, can be removed, opening new avenues for analog design. Efforts are now underway to further evaluate 7a as a preclinical entry for applications to leukemia, with a particular emphasis for CLL patients that display resistance to conventional treatments such as fludarabine.

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EXPERIMENTAL SECTION

Materials and Methods. Chemical reagents were purchased from Acros, Fluka, Sigma−Aldrich, or TCI. Deuterated NMR solvents were purchased from Cambridge Isotope Laboratories. All oligonucleotides 6579

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were custom-synthesized by Integrated DNA Technologies (Skokie, IL, USA). Fludarabine phosphate was purchased from Sigma-Aldrich. All chemical reactions were conducted with rigorously dried anhydrous solvents that were obtained by passing through a solvent column composed of activated A1 alumina. Reactions were conducted under positive pressure of Ar in oven−dried glassware sealed with septa, with stirring from a Teflon coated stir bar using an IKAMAG RCT−basic mechanical stirrer (IKA GmbH). Solutions were heated using either a sand or silicon oil bath. Analytical thin layer chromatography (TLC) was performed on Silica Gel 60 F254 precoated glass plates (EM Sciences). Preparative TLC (pTLC) was conducted on Silica Gel 60 plates (EM Sciences). Visualization was achieved with UV light or an appropriate stain (I2 on SiO 2, KMnO4 , bromocresol green, dinitrophenylhydrazine, ninhydrin, and ceric ammonium molybdate). Flash chromatography was carried out on Geduran Silica Gel 60 (40− 63 mesh) from EM Biosciences. Yields and characterization data correspond to isolated, chromatographically and spectroscopically homogeneous materials. 1H NMR and 13C NMR spectra were recorded on a Varian VX500 spectrometer equipped with an Xsens Cold probe. Chemical shift δ values for 1H and 13C spectra are reported in parts per million (ppm) relative to these referenced values, and multiplicities are abbreviated as s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. All 13C NMR spectra were recorded with complete proton decoupling. FID files were processed using MestraNova 8.0.0 (MestreLab Research). Electrospray (ESI) mass spectrometric analyses were performed using a ThermoFinnigan LCQ Deca spectrometer, and high−resolution analyses were conducted using a ThermoFinnigan MAT900XL mass spectrometer with electron impact (EI) ionization. A Thermo Scientific LTQ Orbitrap XL mass spectrometer was used for high−resolution electrospray ionization mass spectrometry analysis (HR-ESI−MS). All tested compounds possessed a purity of not less than 98% as confirmed by LC−MS analyses prior to use. ((1S,2S)-2-((2S,3S)-3-Methoxypentan-2-yl)cyclopropyl)-methanol (9). A flame-dried 100 mL round-bottom flask was charged with CH2Cl2 (5 mL) and dimethoxyethane (766 uL, 7.36 mmol) and cooled to −10 °C. Et2Zn (13.4 mL, 7.36 mmol) was added in one portion, followed by the addition of CH2I2 (3.94 g, 14.72 mmol) in a dropwise fashion over 20 min. After stirring for an additional 15 min, a solution of dioxaborolane ligand 11 (426 mg, 4.3 mmol) in CH2Cl2 (2 mL) was added dropwise over 5 min. After 10 min, this was followed by the addition of allylic alcohol 8 (292.0 mg, 1.84 mmol) in CH2Cl2 (5 mL) over 5 min. The reaction was allowed to warm to rt over 8 h, at which point it was quenched by the addition of sat. NH4Cl (3 mL) and 10% HCl (10 mL). The mixture was diluted with Et2O (10 mL), the layers were separated, and the aqueous layer was extracted further with Et2O (10 mL). The combined organic layers were transferred to an Erlenmeyer flask and a solution containing 2N NaOH (15 mL) and 30% aqueous H2O2 (3 mL). The resulting biphasic mixture was stirred vigorously for 5 min. The layers were separated, and the organic layer was washed sequentially with 10% HCl (5 mL), sat. NaHCO3 (5 mL), H2O (5 mL), and brine (5 mL); dried over Na2SO4; filtered; and concentrated under reduced pressure to give the crude product. Purification by flash chromatography with 8:1 hexanes/EtOAc afforded cyclopropanol 9 (263 mg, 83%) as a clear oil. 1H NMR (500 MHz, CDCl3) δ 3.37 (dd, J = 6.9, 11.2 Hz, 1H), 3.32 (dd, J = 7.0, 11.2 Hz, 1H), 3.30 (s, 3H), 2.92 (qt, J = 1.9, 5.4 Hz, 1H), 1.51 (td, J = 7.3, 13.9 Hz, 1H), 1.41 (dqd, J = 5.4, 7.4, 14.9 Hz, 1H), 0.86 (m, 3H), 0.80 (t, J = 7.4 Hz, 3H), 0.75 (m, 1H), 0.52 (m, 1H), 0.33 (m, 2H). 13 C NMR (125 MHz, CDCl3) δ 128.3, 86.8, 66.8, 58.0, 40.0, 23.9, 21.0, 20.2, 14.6, 10.3, 10.0. HR-ESI-MS m/z for C10H20O2Na [M]+: calcd. 195.1356, found 195.1341. (1S,2R)-2-((2S,3S)-3-Methoxypentan-2-yl)cyclopropane-1-carbaldehyde (10). Dess−Martin periodinane (634.0 mg, 1.50 mmol) was added to a solution of cyclopropanol 9 (198.0 mg, 1.15 mmol) in CH2Cl2 (20 mL) cooled to 0 °C. After addition, the mixture was warmed to rt. The reaction went to complete after an additional 15 min at rt. At this point, sat. NaHCO3 (4 mL) and sat. Na2S2O3 (4 mL) were added, and the mixture was stirred for 1 h. The layers were separated, and the aqueous layer was further extracted with CH2Cl2

(10 mL). The combined organic layers were washed with sat. NaHCO3 (5 mL), H2O (5 mL), and brine (5 mL); dried over Na2SO4; filtered; and concentrated under reduced pressure. Purification with flash column chromatography using 2:1 hexanes/EtOAc provided pure aldehyde 10 (159.0 mg, 80%) as a clear oil. 1H NMR (500 MHz, CDCl3) δ 8.99 (d, J = 5.6 Hz, 1H), 3.37 (s, 3H), 2.99 (ddd, J = 4.1, 5.5, 7.2 Hz, 1H), 1.62 (tdd, J = 4.3, 5.6, 8.3 Hz, 1H), 1.57 (td, J = 7.3, 13.9 Hz, 1H), 1.48 (m, 2H), 1.30 (td, J = 4.6, 9.1 Hz, 1H), 1.10 (dqd, J = 4.1, 6.8, 9.3 Hz, 1H), 1.03 (ddd, J = 4.7, 6.7, 8.0 Hz, 1H), 0.94 (d, J = 6.9, 3H), 0.88 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz, CDCl3) δ 201.2, 86.5, 58.3, 39.7, 29.7, 26.6, 23.9, 14.7, 14.5, 10.4. HR-ESI-MS m/z for C10H19O2 [M]+: calcd. 171.1380, found 171.1383. (1R,2R)-1-((1S,2R)-2-((2S,3S)-3-Methoxypentan-2-yl)cyclopropyl)2-methylbut-3-yn-1-ol (13a). A solution was prepared containing aldehyde 10 (50.0 mg, 0.29 mmol) and m-allenylstannane 12 (152.0 mg, 0.44 mmol) in CH2Cl2 (6 mL). After cooling to −78 °C, BF3· Et2O (0.11 mL, 0.88 mmol) was added dropwise over 5 min. After stirring for 1 h, a mixture of MeOH (1.6 mL) and sat. NaHCO3 (0.3 mL) was added, and the solution was warmed. After reaching rt, the organic layer was collected. and the aqueous layer was further extracted with CH2Cl2 (5 mL). The combined organic layers were washed with brine (3 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure. Purification by flash column chromatography eluting with 5:1 hexanes/EtOAc provided a mixture of alkynes 13a and 13b (51 mg, 80%). Repeated column chromatography eluting with 10:1 hexanes/EtOAc could separate the two isomers. Alkyne 13a: 1H NMR (500 MHz, C6D6) δ 3.23 (s, 3H), 2.88 (dt, J = 4.4, 6.3 Hz, 1H), 2.62 (m, 1H), 2.57 (m, 1H), 1.83 (d, J = 2.4 Hz, 1H), 1.59 (pd, J = 7.2, 14.5 Hz, 1H), 1.42 (m, 1H), 1.39 (m, 1H), 1.22 (d, J = 7.0 Hz, 3H), 1.09 (d, J = 6.8 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H), 0.77 (m, 2H), 0.61 (dddd, J = 8.5, 9.3, 9.4, 9.8 Hz, 1H), 0.47 (m, 1H), 0.30 (td, J = 4.8, 8.2 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 86.6, 85.3, 78.5, 71.0, 58.3, 40.8, 33.1, 24.0, 23.3, 21.3, 18.0, 14.8, 10.8, 10.4. HR-ESI-MS m/z calcd. for C14H24O2Na [M]+: 247.1669, found 247.1673. Alkyne 13b: 1H NMR (500 MHz, C6D6) δ 3.22 (s, 3H), 2.88 (dt, J = 4.4, 6.3 Hz, 1H), 2.80 (ddd, J = 4.1, 5.7, 7.5 Hz, 1H), 2.60 (ddq, J = 2.5, 5.8, 7.0 Hz, 1H), 1.87 (d, J = 2.5 Hz, 1H), 1.61 (dqd, J = 6.5, 7.4, 13.8 Hz, 1H), 1.47 (d, J = 4.7 Hz, 1H), 1.41 (dqd, J = 6.0, 7.5, 13.6 Hz, 1H), 1.21 (d, J = 7.0 Hz, 3H), 1.03 (d, J = 6.9 Hz, 3H), 0.85 (t, J = 7.4 Hz, 3H), 0.83 (m, 2H), 0.67 (ddd, J = 4.9, 8.7, 10.2 Hz, 1H), 0.37 (td, J = 4.8, 8.3 Hz, 1H), 0.33 (ddd, J = 4.7, 5.5, 8.5 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 86.8, 86.3, 78.3, 70.4, 58.1, 39.7, 33.1, 23.9, 21.1, 20.5, 16.7, 14.4, 10.9, 10.4. HR-ESI-MS m/z for C14H24O2Na [M]+: calcd. 247.1669, found 247.1674. (2S,3S,6S,7R,10R,E)-7,10-Dihydroxy-2-((2E,4E,6R,7S)-7-hydroxy-7((1S,2R)-2-((2S,3S)-3-methoxypentan-2-yl)cyclopropyl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl Acetate (7a). Compound 7a was prepared via a two-step procedure that began with the hydrostannation of 13a to vinylstannane 14a, followed by Stille coupling of 14a to vinyl iodide 15. This procedure was best conducted in a rapid fashion without storing 14a. PdCl2(PPh3)2 (2.1 mg, 3.0 μmol) was added to a solution of alkyne 13a (14.0 mg, 62.0 μmol) in THF (5 mL) cooled to 0 °C. This was followed by the addition of n-Bu3SnH (35 uL, 0.127 mmol) in a dropwise fashion over 5 min. After stirring for 30 min, the reaction mixture was concentrated under reduced pressure and quickly run through a short silica gel column eluting with 15:1 hexanes/EtOAc containing 2% Et3N to give the crude vinyl stannane 14a (16.0 mg). Immediately after preparation, Pd2(dba)3 (0.4 mg, 0.4 μmol), Ph3As (3 mg, 8.8 μmol), and LiCl (0.5 mg, 13 μmol) were added sequentially to a solution of vinyl iodide 15 (2 mg, 4.2 μmol) and stannane 14a (2.2 mg, 4.2 μmol) in dried and degassed N-methylpyrrolidone (NMP) (0.3 mL). The reaction was stirred for 24 h, at which point an additional Pd2(dba)3 (0.4 mg, 0.4 μmol) and Ph3As (3.0 mg, 8.8 μmol) were added. After an additional 24 h at rt, H2O (1 mL) was added, and the mixture was extracted with EtOAc (3 × 5 mL). The combined organic layers were washed with H2O (5 mL) and brine (5 mL), dried with Na2SO4, and concentrated. The remaining NMP was removed under a stream of N2. Flash chromatography with a gradient from 1:1 hexanes/EtOAc to 2:1 EtOAc/hexanes, followed by pTLC in 6580

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Apoptosis Induction Assay. Apoptotic and viable cells were discriminated via flow cytometry of cells stained with 3,3′dihexyloxacarbocyanine iodide (DiOC6) (Molecular Probes, Eugene, OR, USA) and propidium iodine (PI) (Sigma, St Louis, MO, USA). Using this method, viable cells exclude PI and stain brightly positive for DiOC6. RT-PCR Splicing Assay. B-CLL cells were treated with 100 nM 1a, 100 nM 1b, 10 μM fludarabine (F-Ara-A), and a varied concentration of 7b (100−1000 nM) for 4 h. Total RNA was extracted using mirVana miRNA isolation kit (Life Technologies, Grand Island, NY USA). After DNase treatment, 200 ng of total RNA was reverse-transcribed using a SuperScript III first-strand synthesis system (Life Technologies, Grand Island, NY, USA). PCR was performed on 10 ng of the obtained cDNA or human genomic DNA as a control in 20 μL of reaction mixture containing Plantinum PCR SuperMix High-fidelity (Life Technologies, Grand Island, NY, USA) and 0.1 μM of each primer of the appropriate pair. PCR conditions were 94 °C for 3 min; 35 cycles of 94 °C for 30 s, annealing temperature (58 and 55 °C for DNAJB1 and GAPDH, respectively) for 30 s, and 72 °C for 1 min; followed by 72 °C for 5 min. PCR products were separated on a 2% agarose gel and stained with ethidium bromide. In Vitro Stability Assays. Compounds were dissolved in NMR grade solvents and stored at 37 °C for the ascribed period. NMR analyses were collected at 500 MHz on a Joel ECA500 NMR machine or VX500 NMR machine equipped with an X-Sens probe.

a chamber containing 2:1 EtOAc/hexanes, afforded isomer 7a (1.7 mg, 73%), a white wax. 1H NMR (500 MHz, C6D6) δ 6.40 (ddd, J = 1.0, 10.9, 15.2 Hz, 1H), 6.24 (dd, J = 1.6, 10.9, Hz, 1H), 5.98 (dd, J = 8.7, 15.2 Hz, 1H), 5.82 (dd, J = 9.7, 15.3 Hz, 1H), 5.62 (dd, J = 9.9, 15.3 Hz, 1H), 5.29 (d, J = 10.6 Hz, 1H), 5.25 (d, J = 9.7 Hz, 1H), 3.63 (d, J = 11.0 Hz, 1H), 3.48 (m, 1H), 3.24 (s, 3H), 2.88 (dt, J = 3.6, 6.3 Hz, 1H), 2.67 (dd, J = 3.6, 8.0 Hz, 1H), 2.55 (ddd, J = 4.0, 7.3, 7.4 Hz, 1H), 2.42 (m, 3H), 2.22 (dd, J = 4.0, 15.0 Hz, 1H), 2.17 (dd, J = 3.1, 14.9 Hz, 1H), 1.67 (d, J = 1.2 Hz, 3H), 1.62 (s, 3H), 1.57 (m, 1H), 1.41 (s, 1H), 1.35 (m, 3H), 1.18 (d, J = 6.9 Hz, 3H), 1.06 (d, J = 6.3 Hz, 3H), 1.01 (s, 3H), 0.92 (m, 2H), 0.84 (t, J = 7.4 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H), 0.65 (m, 2H), 0.34 (td, J = 4.5, 7.3 Hz, 1H), 0.20 (td, J = 4.8, 7.7 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 172.1, 169.0, 140.7, 138.8, 131.9, 130.9, 126.5, 126.4, 86.5, 82.7, 79.4, 79.2, 73.3, 69.3, 57.8, 43.9, 41.2, 41.1, 38.5, 35.9, 24.8, 24.2, 23.4, 21.8, 20.8, 18.0, 16.5, 15.2, 11.8, 10.4, 10.3, 1.4. HR-ESI-MS m/z for C32H52O8Na [M]+: calcd. 587.3554, found 587.3553. (2S,3S,6S,7R,10R,E)-7,10-Dihydroxy-2-((2E,4E,6R,7S)-7-hydroxy-7((1S,2R)-2-((2S,3S)-3-methoxypentan-2-yl)cyclopropyl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl Acetate (7b). PdCl2(PPh3)2 (1.7 mg, 2.4 μmol) was added to a solution of alkyne 13b (11.0 mg, 49.0 μmol) in THF (5 mL) cooled to 0 °C. This was followed by the addition of n-Bu3SnH (27 μL, 101.0 μmol) in a dropwise fashion over 5 min. After stirring for 30 min, the reaction was concentrated under reduced pressure and quickly run through a short silica gel column eluting with 15:1 hexanes/EtOAc with 2% Et3N to afford the crude 14b (18 mg, 73%). Immediately after preparation, Pd2(dba)3 (0.4 mg, 0.4 μmol), Ph3As (3 mg, 8.8 μmol), and LiCl (0.5 mg, 13 μmol) were added sequentially to a solution of vinyl iodide 15 (2 mg, 4.2 μmol) and the crude stannane 14b (2.2 mg, 4.2 μmol) in dried and degassed NMP (0.3 mL). The reaction was stirred for 24 h, at which point an additional Pd2(dba)3 (0.4 mg, 0.4 μmol) and Ph3As (3 mg, 8.8 μmol) were added. After an additional 24 h at rt, H2O (1 mL) was added, and the mixture was extracted with EtOAc (3 × 5 mL). The combined organic layers were washed with H2O (5 mL) and brine (5 mL), dried with Na2SO4, and concentrated. The remaining NMP was removed under a stream of N2. Flash chromatography with a gradient from 1:1 hexanes/EtOAc to 2:1 EtOAc/hexanes, followed by pTLC in a chamber containing 2:1 EtOAc/hexanes, afforded isomer 7b (1.9 mg, 80%), a white wax. 1H NMR (500 MHz, CDCl3) δ 6.37 (ddd, J = 1.1, 10.8, 15.2 Hz, 1H), 6.23 (dd, J = 1.5, 10.8 Hz, 1H), 5.85 (dd, J = 7.9, 14.8 Hz, 1H), 5.81 (dd, J = 9.6, 15.3 Hz, 1H), 5.62 (dd, J = 10.0, 15.2 Hz, 1H), 5.29 (d, J = 10.7 Hz, 1H), 5.24 (d, J = 9.8 Hz, 1H), 3.62 (d, J = 11.1 Hz, 1H), 3.48 (m, 1H), 3.21 (s, 3H), 2.86 (dt, J = 4.2, 6.3 Hz, 1H), 2.67 (dd, J = 5.6, 8.1 Hz, 1H), 2.46 (dq, J = 1.2, 6.9 Hz, 1H), 2.39 (m, 1H), 2.25 (dd, J = 3.9, 14.9 Hz, 1H), 2.17 (dd, J = 3.0, 14.9 Hz, 1H), 2.10 (m, 1H), 1.68 (d, J = 1.3 Hz, 3H), 1.61 (s, 3H), 1.56 (m, 1H), 1.36 (s, 1H), 1.29 (m, 4H), 1.18 (d, J = 6.85 Hz, 3H), 1.00 (s, 3H), 0.96 (d, J = 6.8 Hz, 3H), 0.91 (m, 2H), 0.83 (t, J = 7.4 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H), 0.60 (m, 2H), 0.27 (d, J = 6.8 Hz, 2H). 13C NMR (125 MHz, CDCl3) δ 172.1, 169.0, 140.7, 139.8, 131.9, 131.0, 126.4, 125.5, 86.6, 82.7, 79.4, 79.2, 73.3, 69.3, 57.6, 44.2, 41.2, 40.1, 38.5, 35.8, 32.4, 24.8, 24.0, 22.6, 21.1, 20.7, 16.5, 16.2, 14.5, 11.9, 10.7, 10.4, 1.4. HR-ESI-MS m/z for C32H52O8Na [M]+: calcd. 587.3554, found 587.3552. Chronic Lymphocytic Leukemia Samples and Cell Culture Conditions. Chronic lymphocytic leukemia samples were obtained from the CLL Research Consortium (CRC) tissue bank. The blood samples were collected after obtaining a written informed consent from the patients under a protocol approved by the institutional review board of The University of California, San Diego. All patients met criteria for CLL. The peripheral blood mononuclear cells were separated from heparinized venous blood of the CLL patients by density gradient centrifugation using Ficoll−Hypaque separation (GE Healthcare, USA). The samples that were used had more than 95% positivity for B cells confirmed by staining using CD19 and CD5+, followed by flow cytometry. CLL B cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 U/mL of penicillin and 100 μg/mL of streptomycin at 37 °C in an atmosphere of 5% CO2.

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S Supporting Information *

Selected copies of 1H NMR, 13C NMR, and 1H−1H gCOSY NMR spectra for compounds 7a, 7b, 9, 10, 13a, and 13b as well as NCI-60 data collected from analyses of 5a, 6a, and 6c. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone +1 858-534-5673. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was support by financial support from the American Cancer Society (RSG-06-011-01-CDD), NIH (R01GM086225), and a Lymphoma Research Foundation Grant (No. 285871). We thank Dr. Yongxuan Su (UC San Diego) for mass spectral analyses, and Drs. Anthony Mrse and Xuemei Huang (UC San Diego) for assistance with acquiring NMR spectral data.

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ABBREVIATIONS USED CLL, chronic lymphocytic leukemia; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; DNAJB1, DnaJ (Hsp40) homologue subfamily B member 1; F-Ara-A, fludarabine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HR-ESI-MS, high-resolution electrospray ionization mass spectrometry analysis; MOA, mode of action; PBMCs, peripheral blood mononuclear cells; PI, propidium iodine

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REFERENCES

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